Butanol is an important bulk chemical with a wide range of industrial uses that has worldwide production of 4.5-5.5 million tonnes per annum. It is used as a precursor for the production of acrylate and methacrylate esters (used in coatings, plastics, textiles, adhesives, etc), glycol ethers (coatings, electronics) and butyl acetate (paints, ink, coatings, synthetic fruit flavoring) as well as butylamines (production of pesticides and pharmaceuticals) and amine resins. It also has direct use as a solvent (in ink, dyes, etc), an extractant (for the production of drugs and natural substances such as alkaloids, antibiotics, hormones, and vitamins), and in deicing fluids, cosmetics and chromatography.
Butanol also has potential as a second generation biofuel, and in this context is referred to as Biobutanol (Köpke & Dürre, 2010). It has similar properties to gasoline and superior properties to ethanol. Specifically, it has increased mileage due to higher energy density, it can be mixed with gasoline in any concentration (while ethanol can only be blended up to 85%) and is not hygroscopic or corrosive.
Biofuels for transportation are attractive replacements for gasoline and are rapidly penetrating fuel markets as low concentration blends. Biofuels, derived from natural plant sources, are more environmentally sustainable than those derived from fossil resources (such as gasoline), their use allowing a reduction in the levels of so-called fossil carbon dioxide (CO2) gas that is released into the atmosphere as a result of fuel combustion. In addition, biofuels can be produced locally in many geographies, and can act to reduce dependence on imported fossil energy resources.
The vast majority of biofuels are produced via traditional yeast-based fermentation processes that use crop derived carbohydrates as the main carbon source and are known as first generation biofuels. However, these crops are required for food and many crops also require high agricultural inputs in the form of fertilizers. These limitations mean that first generation biofuels are considered unsustainable and the greenhouse gas reductions that can be achieved are limited. The aim of second generation biofuels is the sustainable use of non-food parts of current crops or other industrial waste to reduce greenhouse gas emissions and reduce dependency on fossil fuels.
Recent 1-butanol production has been mainly by oxo synthesis (Weiβermel & Arpe, 2003). Petrochemicals including crude oil are cracked to form propylene which is used during oxo synthesis. However the synthesis process requires use of non-renewable resources as well as suffering from being expensive and non-specific in the products formed.
Butanol can also be produced through biological production methods, the most common being the Acetone-Butanol-Ethanol (ABE) fermentation which has been used industrially since 1913 (Köpke & Dürre, 2010). This method has the unwanted by-product of acetone which is usually produced at about half the volume of butanol which therefore substantially reduces the yield. Additionally, this method of fermentation is limited by the toxicity of butanol to the micro-organism which results in growth being almost completely inhibited at such low butanol concentrations as 1.5% (Köpke and Dürre 2010). Furthermore ABE fermentation uses sugar from corn, starch, cassava and sugar cane as a feedstock. This results in the undesirable use of arable land to produce fuel rather than food. It can also exacerbate problems related to deforestation and desertification.
Only a few organisms are known to naturally produce butanol and none of these produce butanol at a high yield from abundant sources (such as carbon monoxide—CO). Two organisms known to naturally produce butanol from CO are Butyribacterium methylotrophicum (which synthesises only traces of butanol (Heiskanen et al, 2007)), and Clostridium carboxidivorans (which produces low yields of 1-butanol as a by-product to the main fermentation products ethanol and acetate (Liou et al, 2005)).
A number of organisms have been genetically modified to produce 1-butanol including E. coli, Bacillus subtilis, Saccharomyces cerevisiae, Pseudomonas putida, or Lactobacillus brevis. However all of these organisms still rely on sugar as feedstock (Köpke & Dürre, 2010). Despite over 250 Clostridium species being known, only a few are genetically accessible. There is no natural competence (uptake of extracellular DNA from the cell's environment) known in Clostridia and electrotransformation or conjugation are the only methods available for transformation. These issues present significant difficulties in effectively transforming Clostridia species. Most Clostridia have one or more restriction/methylation systems to protect against foreign and phage DNA which means that transformation is particularly difficult and unpredictable.
Bibliographic details of the publications referred to herein are collected at the end of the description.
It is an object of the invention to overcome one or more disadvantages of the prior art, or to at least provide the public with a useful alternative to known technologies.